. Septic shock occurs in more that 500,000 patients each year in the United States, nearly half of who die. Activation of macrophages by microbial products such as Gram-negative bacterial lipopolysaccharide (LPS) leads to the release of inflammatory mediators that are a major factor in the systemic inflammatory response of sepsis. LPS binds to several macrophage membrane receptors, which activate multiple signaling cascades. Potential interactions of the signal transduction pathways activated by LPS via its receptors, and post-receptor transducers, remain to be clearly defined. Post-receptor coupling to specific guanine nucleotide regulatory (G) proteins constitutes a novel pathway of LPS activation. These signal transduction pathways may be distinct from LPS signaling events leading to nuclear translocation of the transcription factor NFB. LPS tolerance induced by pre-exposure to low concentrations of LPS increases resistance to LPS lethality and alters Mi signal transduction and mediator release. Altered macrophage signal transduction is a hallmark of tolerance. In LPS tolerant macrophage, G protein-coupled signaling pathways are altered. The specific LPS receptors or signal transduction pathways that induce tolerance are not known. However induction of LPS tolerance has been linked to changes in the NFB p50 subunit composition. The major hypothesis is that LPS tolerance alters G protein regulated signal transduction pathways through NFB initiated transcription-dependent events. Specific aims proposed to test this hypothesis will: 1) determine Galpha protein regulated pathways of activation by LPS and their alterations in LPS tolerance and 2) determine the role of NFkB-initiated transcription events in LPS tolerance as a mechanism for altered G protein signal transduction. In the first Specific Aim genetic, pharmacologic, and molecular approaches will delineate potential links between specific LPS receptors and G protein regulated pathways activated by LPS. Proximal signal changes in the G protein coupled pathways induced by LPS tolerance will be characterized in cell lines, and in primary murine and rat peritoneal macrophages and human monocyte. The effect of inhibiting G protein regulated signaling in murine sepsis models will be tested. Specific Aim 2 will investigate links between NFB and tolerance-induced changes in the G protein coupled signaling. We will use mice genetically deficient in NFB (p50) and cellular over-expression of NFB (p50). Induction of phosphatases by NFB will be examined as a mechanism of altered signal transduction in LPS tolerance. Delineation of cellular mechanisms responsible for LPS tolerance may provide a rational basis for therapeutic interventions in sepsis.